Administration of Topical NorLeu3Angiotensin(1-7) Minimizes Fibrotic Corneal Healing in Stellate Wound: A 28-Day Study

Figures

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  Figure 1. Evaluation of corneal haze. (A,B) Corneal haze scores indicate none (0), trace (0.5), minimal (1), mild (2), moderate (3), or severe (4). Median haze scores are plotted, and error bars represent range. (C) Area of corneal haze in pixels, normalized to corneal area in pixels, demonstrates reduced corneal haze with 0.1% NLE treatment compared with 0.3% NLE (p < 0.05). (D) Representative images of each treatment group on Day 28. ** p < 0.01. * p < 0.05.

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  Figure 2. Corneal confocal microscopy imaging on Day 28. (A) Representative images (400 × 400 μm) from each treatment group are pictured for each layer and represent the keratocyte density throughout the stroma for treatment group. (B) Stromal keratocyte density was average from five HRT-III images from each stroma. Keratocyte levels are significantly higher in the 0.1% NLE treatment group (p < 0.05) than the vehicle. (C) The change in stromal thickness from Day 0 to 28 shows reduced corneal edema with 0.1% NLE (** p < 0.01) and 0.45% NLE (* p < 0.05).

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  Figure 3. Corneal sections stained with Masson’s trichrome to visualize collagen formation (blue) in uninjured (C) and injured stroma (A,B,D,E). Images captured at 40× magnification. Collagen formation plotted as percent area of tissue stained blue (F). Collagen area significantly reduced following injury after treatment with 0.3% and 0.45% NLE. ** p < 0.01. Scale bar = 20 μm.

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  Figure 4. Immunofluorescence staining of αSMA (red) with DAPI (blue). Images captured at 40× magnification in uninjured stroma (C) and injured stroma (A,B,D,E). Area of αSMA stain is reduced with 0.1% and 0.45% NLE compared to 0.3% NLE (F). **** p < 0.0001; ** p < 0.01; * p < 0.05.

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  Figure 5. Immunofluorescence staining of TGFβR1 (red) and TGFβ1 (green) in the corneal stroma, counterstained with DAPI (blue). (A) Representative 40× images of uninjured and injured corneas. (B) Area of TGFβR1 is elevated in injured corneas treated with vehicle or 0.3% or 0.45% NLE. (C) TGFβ1 is somewhat elevated in vehicle-treated corneas. ** p < 0.01. Scale bar = 20 μm.

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  Figure 6. Relative gene expression in injured corneas. Fold change is in comparison to vehicle and expressed as 2−ΔΔCt. Targets evaluated include components related to the RAS pathway (A), TGFβ-Smad-dependent pathway (B), and collagen formation (C).